Resorbable macroporous bioactive glass scaffold and method of manufacture

ABSTRACT

A method of manufacturing a resorbable, macroporous bioactive glass scaffold comprising approximately 24-45% CaO, 34-50% SiO 2 , 0-25% Na 2 O, 5-17% P 2 O 5 , 0-5% MgO and 0-1% CaF 2  by mass percent, produced by mixing with pore forming agents and specified heat treatments.

This application is a continuation application of U.S. patentapplication Ser. No. 12/798,660, filed Apr. 6, 2010, which is adivisional application of and claims the benefit of U.S. patentapplication Ser. No. 11/329,469, filed Jan. 11, 2006 now issued U.S.Pat. No. 7,758,803, contents of which are incorporated herein in theirentirety.

TECHNICAL AREA

This invention relates to the area of biomaterials involving resorbableor degradable, macroporous bioactive glass material which can be usedeither for the restoration of hard tissues or as the tissue engineeringscaffold, as well as preparation methods for such materials.

BACKGROUND TECHNOLOGY

There has been a history of over 30 years in research on bioactive glasssince 1971 when Dr. Larry Hench reported that such glass could bondtogether with bone tissues for the first time. Also, such glass materialhas been used for restoration of bone defects in clinical practice forover ten years, and such clinical applications have proven successful inthat this glass can bring along not only the benefit of osteoconduction,but also the bioactivity to stimulate the growth of bone tissues. Manyrecent studies have revealed that the degradation products of bioactiveglass can enhance the generation of growth factors, facilitate cellularproliferation and activate gene expression of osteoblasts. Moreover,bioactive glass is the only synthetic biomaterial so far that can bothbond with bone tissues and soft tissues. These unique features of thisglass have created a great potential for its clinical application as atype of medical device, and thereby, attracted great attention from bothacademia and the industrial sector. Despite its excellentbiocompatibility and bioactivity, bioactive glass can be now producedonly in a granular form for clinical application. For restoration ofbone defects, macroporous and block scaffold materials with a particularmechanical strength are often needed to fill in and restore suchdefects. Even in the field of tissue engineering, which receivesworld-wide attention and evolves rapidly, macroporous bioactive scaffoldmaterials are similarly demanded to serve as cell carriers.

Research studies in the past have suggested that besides the compositionof the material, its structure can directly influence its clinicalapplications as well. The macroporous and block scaffold materials withbioactivity whose pore sizes are in the range of 50-500 microns are mostsuitable to be used as materials either for the restoration of bonedefects, or as cell scaffolds. Any macroporous biomaterial having a poresize within the said range can bring benefits to the housing andmigration of cells or tissue in-growth, as well as to the bonding ofsuch a material to living tissues, thereby achieving the goals ofrepairing defects in human tissues and reconstructing such tissues moreeffectively.

Moreover, the subject of the biomaterials that are both resorbable andmacroporous has now become an integral part of tissue engineeringstudies that have been rapidly developed in recent years, wherescaffolds made of such macroporous materials can be adopted to serve ascell carriers so that cells can grow in the matrix materials andconstitute the living tissues that contain genetic information of thecell bodies, and such tissues can be in turn, implanted into humanbodies to restore tissues and organs with defects. Therefore,resorbable, macroporous bioactive glass scaffold materials possesswide-ranging potential for their applications as cell scaffolds eitherfor restoration of defects in hard tissues, or for the purpose of invitro culture of bone tissues.

U.S. Pat. Nos. 5,676,720 and 5,811,302 to Ducheyne, et al, teach ahot-pressing approach using inorganic salts such as calcium carbonateand sodium bicarbonate as the pore-forming agents to prepare andmanufacture macroporous bioactive glass scaffolds which have thecompositions of CaO—SiO₂—Na₂O—P₂O₅, and which are designed to functionas the cell scaffolds used for in vitro culture of bone tissues.Nevertheless, this hot-pressing approach if adopted would entail highproduction costs, and furthermore, controlling the composition of thefinished products is difficult because the composition will be affectedby the remnants that result after sintering the inorganic salts used aspore-forming agents. Additionally, Yuan, et al. have adopted oxydol as afoaming agent to prepare and manufacture 45S5 bioactive glass scaffoldsunder a temperature of 1000° C., with the scaffolds produced in this waybeing bioactivity and having the ability to bond together with bonetissues (J. Biomed. Mater. Res; 58:270-267, 2001). But according to ourtesting results, the glasses will become substantially crystallized andtheir resorbability/degradability will decrease if they are sinteredunder a temperature of 1000° C. In addition, it is quite difficult tocontrol the pore size and pore number of the materials when oxydol isused as the foaming agent.

Mechanical strength is also an important factor for performance ofmacroporous bioactive glass scaffold materials, and relevant studieshave suggested that any compressive strength below 1 MPa would result inthe poor applicability of these scaffold materials, and thus, in thecourse of applying them either as cell scaffolds or for the purpose ofrestoration of bone injuries, such materials would be very prone tobreakage or damage, therefore limiting the effectiveness of theirapplication. So far, no report on the compressive strength standard dataof macroporous bioactive glass scaffolds has been found in previouspatent and published documents and as a result, gives rise to thepurpose of this invention to determine proper technical control measuresto keep the compressive strength of the manufactured bioactive glassscaffold within a certain range to meet the requirements of variousapplications.

SUMMARY OF THE INVENTION

The purpose of this invention is to develop, through the optimization oftechnology and process, a new type of macroporous bioactive glassscaffold with interconnected pores, which features excellentbioactivity, biodegradability, controllable pore size and porosity. Sucha scaffold would serve as a means to repair defects in hard tissues andbe applied in the in vitro culture of bone tissues, and its strength canbe maintained within a range of 1-16 MPa in order to meet demandsarising from the development of the new-generation biological materialsand their clinical applications.

This invention has been designed to use glass powders as raw material,into which organic pore forming agents will be added, and the mixturewill be processed by either the dry pressing molding method orgelation-casting method, and then the resulting products will beobtained by sintering under appropriate temperatures. In this way, amacroporous bioactive glass scaffold can be obtained with variousporosities, pore sizes and pore structures, as well as different degreesof compressive strength and degradability. The chemical composition ofsuch scaffolds shall be expressed as CaO 24-45%, SiO₂ 34-50%, Na₂O0-25%, P₂O₅ 5-17%, MgO 0-5 and CaF₂ 0-1%. Additionally, the approachesprovided in this invention can be adopted to prepare the said scaffoldin different shapes. The crystallizations of calcium phosphate and/orcalcium silicate can be formed inside the bioactive glass scaffolds byway of technical control, whereby both the degradability and mechanicalstrength of the macroporous materials can be controlled as demanded.

As designed in this invention, the macroporous bioactive glass scaffoldmaterials exhibit excellent biological activity, and can release solublesilicon ions with precipitation of bone-like hydroxyl-apatitecrystallites on their surface in just a few hours after being immersedinto simulated body fluids (SBF). In addition, the macroporous bioactiveglass in this invention is resorbable, as shown by in vitro solubilityexperiments, and such glass demonstrates a degradation rate ofapproximately 2-30% after being immersed in simulated body fluids (SBF)for 5 days. As such, it can be concluded that the macroporous bioactiveglass scaffold materials in this invention do not only have desirablebiointerfaces and chemical characteristics, but also demonstrateexcellent resorbability/degradability.

Another feature of this invention is manifested in controlling technicalconditions to create materials that can have both a relatively higherporosity (40-80%) with suitable pore size (50-600 microns), and exhibita proper mechanical strength (with the compressive strength at 1-16MPa).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photograph of the prepared macroporous bioactive glass.

FIG. 2 is an optical microscope picture displaying cross-sections of themacroporous bioactive glass.

FIG. 3 shows XRD displays for the macroporous bioactive glass materialsprepared under different temperatures; these illustrations show thatdifferent levels of crystallization of calcium silicate or calciumphosphate can be found on the surface of the materials prepared underdifferent temperatures; (a) bioactive glass powder before sintering, (b)bioactive glass scaffolds prepared by sintering at 800° C., (c)bioactive glass scaffolds prepared by sintering at 850° C.

FIG. 4(A) is an SEM picture of the macroporous bioactive glass materialof this invention before being immersed in SBF (i.e. simulated bodyfluids); 4(B) is an SEM picture of the material immersed SBF for 1 day;4(C) is an SEM picture of the material when immersed in SBF for over 3days; these pictures show that substantial hydroxyapatite crystallinecan form on the surface of the material when immersed in SBF for 1 day.

FIG. 5 is a Fourier Transform Infrared spectrometry (FTIR) spectra ofthe macroporous bioactive glass materials before being immersed in SBF,as well as after being immersed in SBF for 0 hours, 6 hours, 1 day, 3days and 7 days respectively; the resulting analysis reveals that thehydroxyl-apatite peak can be observed when such material has beenimmersed in SBF for only 6 hours.

DETAILED DESCRIPTION OF THE INVENTION

The implementation of this invention is detailed as below:

1. Preparation of Materials:

The bioactive glass powder in this invention is prepared using themelting method. The inorganic materials applied in the present inventionare all of analytical purity. Specifically, these chemical reagents areweighed and evenly mixed in line with requirements for propercomposition results, and then melted in temperatures ranging from 1380°C. to 1480° C. to produce glass powders with a granularity varying from40 to 300 μm after cooling, crushing and sieving procedures.Furthermore, such glass powders are then used as the main raw materialto prepare a variety of the macroporous bioactive glass scaffoldsubstances by way of different processing technologies. The pore formingagents specified in the present invention can be organic or polymermaterials such as polyethylene glycol, polyvinyl alcohol, paraffin andpolystyrene-divinylbenzene, etc., whose granularity can fall in therange of 50-600 microns. Thus, the pore forming agent within a certaingranularity range (20-70% in mass percent) can be blended with the saidbioactive glass powders and the resulting mixture can be molded byadopting either of the following two approaches:

First, the dry pressing molding approach, in which 1-5% polyvinylalcohol (concentration at 5-10%) is added to the said mixture as theadhesive, which is stirred, and then dry-pressed into a steel mold(pressure at 2-20 Mpa) to produce a pellet of the macroporous material,which is then sintered (temperature at 750-900° C.) for 1-5 hours toobtain final product.

Second, the gelation-casting approach, in which an aqueous solution isprepared as per the following mass percent concentrations: 20%acrylamide, 2% N, N′-methylene-bis-acrylamide cross-linking agents and5-10% polyacrylic acid dispersant agents. Next, the aforementionedmixture and the aqueous solution (volume percent at 30-60%) is combinedand mixed, and ammonium persulfate (1-5% in mass percent) and N, N,N′,N′-tetramethyl ethylene diamine (1-5% in mass percent) is added. Then,the above-mentioned materials are stirred to produce a slurry with finefluidity and homogeneity, which is then poured into plastic or plastermolds for gelation-casting. Later the cross-linking reaction of monomersis induced under temperatures ranging from 30° C. to 80° C. for 1-10hours, and pellets of the macroporous material are obtained after a fewhours of drying at 100° C. The pellets are processed first at thetemperature of 400° C. to remove organics, and then sintered at 750-900°C. to obtain the macroporous material of the present invention.

2. Performance Evaluation

2.1. The Mechanical Strength of the Macroporous Material:

An array of samples obtained in this invention was tested for theirrespective compressive strengths using the Autograph AG-I ShimadzuComputer-Controlled Precision Universal Tester made by the ShimadzuCorporation. The testing speed designated for these samples was 5.0mm/min. This test revealed that the compressive strength of themacroporous material obtained in this invention can be well controlledwithin the scope of 1-16 MPa.

2.2. The Porosity of the Macroporous Materials

The Archimedes Method was used to carry out a test with a part of thesamples mentioned above to determine their porosities, and a ScanningElectron Microscope (SEM) was used to observe their pore shapes anddistribution. This test demonstrated that the porosity of themacroporous material obtained in this invention can be well controlledwithin a range of 40-80%.

2.3 Bioactivity Evaluation

A test of in vitro solution bioactivity was carried out with themacroporous materials obtained in the present invention, after beingwashed in de-ionized water and acetone successively, and then air driedafterwards. The solution applied was simulated body fluids (SBF). Theion and ionic group concentrations in this SBF are the same as those inhuman plasma. This SBF's composition is as below:

-   NaCl: 7.996 g/L-   NaHCO₃: 0.350 g/L-   KCl: 0.224 g/L-   K₂HPO₄.3H₂O: 0.228 g/L-   MgCl₂.6H₂O: 0.305 g/L-   HCl: 1 mol/L-   CaCl₂: 0.278 g/L-   Na₂SO₄: 0.071 g/L-   NH₂C(CH₂OH)₃: 6.057 g/L

The test was carried out with macroporous material immersed in SBF inthe following conditions: 0.15 g of macroporous material, 30.0 ml/daySBF, 37° C. in a temperature-controlled water-bath. After themacroporous material was immersed in SBF for a period of 1, 3 or7 daysrespectively, samples were taken out and washed using ion water, andthen underwent the SEM, Fourier Transform Infrared spectrometry (FTIR)and XRD tests. The respective results of the tests can be seen in FIGS.3, 4 and 5. The relevant bioactivity experiment results have shown thatthe macroporous glass scaffold materials obtained in the presentinvention can induce the formation of bone-like hydroxyapatite on theirsurface, indicating ideal bioactivity of these materials.

2.4 Degradability Evaluation

A bioactivity experimental test was conducted on the macroporousmaterials in this invention after being washed in de-ionized water andacetone successively, and then dried. Evaluation of both degradationspeed and degradability of the macroporous materials according to thecontent of SiO₂ substances that are released at different time pointsafter the materials have been immersed in SBF was conducted. Forexample, where PEG is used as the pore forming agent, the macroporousbioactive glass scaffolds (porosity at 40%) obtained after the processesof dry pressing molding and calcination (temperature at 850° C.) exhibita degradability of 10-20% when the scaffold has been immersed in SBF for5 days.

Implementation Example 1

The raw materials used in this example are the same as those describedabove.

SiO₂, Na₂CO₃, CaCO₃ and P₂O₅ (all of analytical purity) are mixedproportionally, and the mixture is melted into homogenous fused massesat the temperature of 1420° C. and then cooled, crushed and sieved toobtain bioactive glass powder with a particle diameter ranging from40-300 microns. The composition of the bioactive glass powder isexpressed as CaO 24.5%, SiO₂ 45%, Na₂O 24.5% and P₂O₅ 6%. Next, thebioactive glass powder (150-200 microns in granularity) is mixed withthe polyethylene glycol powder (200-300 microns in granularity) at amass percent of 60:40. Polyvinyl alcohol solution (6%), which serves asthe adhesive, is added and the solution is mixed. The mixture is thendry-pressed under a pressure of 14 MPa, and the pellets of themacroporous materials are stripped from the mold. The pellets are firstprocessed at 400° C. to remove organics, and then sintered at 850° C.for 2 hours to obtain the said macroporous materials with a compressivestrength at approx. 1.25 MPa and a porosity at about 56%. The XRDindicates the existence of both the Ca₄P₂O₉ and CaSiO₃, as shown in FIG.2(C).

Finally, the said macroporous materials are immersed in simulated bodyfluids (SBF) for periods of 6 hours and 1, 3, and 7 days respectively,and evaluated as to both bioactivity and resorbability/degradability.Results in FIGS. 4 and 5 demonstrate that the macroporous glass materialof this invention has strong bioactivity, as a bone-like apatite layeris soon formed on the surface of such materials after they are immersedin SBF. After this material has been immersed in SBF for 5 days, itsdegradation rate can be up to a level of 14%, suggesting that themacroporous bioactive glass material in this invention has idealdegradability, and can therefore be expected to be successfully appliedfor the restoration of injured hard tissues and as the cell scaffold forin vitro culture of bone tissue.

Implementation Example 2

SiO₂, CaCO₃, Ca₃ (PO4)₂, MgCO₃, CaF₂ (all of analytical purity) aremixed proportionally, melted into a homogenous fused masses at thetemperature of 1450° C., and then cooled, crushed and sieved to obtainbioactive glass powder (particle diameter ranging from 40-300 microns).The composition of the bioactive glass powder is CaO 40.5%, SiO₂ 39.2%,MgO 4.5%, P₂O₅ 15.5% and CaF₂ 0.3%.

Next, the bioactive glass powder is blended with polyvinyl alcoholpowder (300-600 microns in granularity) at a mass percent of 50:50 toobtain a solid mixture. An aqueous solution composed of 20% acrylamide,2% N,N′-Methylene-bis-acrylamide and 8% polyacrylic acid is prepared,and 10 grams of the said solid mixture is blended with the aqueoussolution at a volume percent (ratio) of 50:50, with several drops ofammonium persulfates (3% in mass percent) and several drops ofN,N,N′,N′-tetramethyl ethylene diamine (3% in mass percent) added andstirred to produce a slurry with fine fluidity, which is poured intomolds for gelation-casting. The cross-linking reaction of monomers ofthe material is induced for 3 hours at 60° C. In this way, pellets ofthe macroporous material are obtained by stripping them from the moldafter the gelation-casts have been dried at 100° C. for 12 hours.Subsequently, the pellets are processed at 400° C. to remove organics,and then sintered at 850° C. for 2 hours to produce the macroporousmaterials that feature a compressive strength at about 6.1 MPa andporosity at approx. 55%. This material demonstrated degradability is 78%(calculated based on the mass percent of Si releasing) after beingimmersed in Simulated Body Fluids for 3 days.

Implementation Example 3

The raw materials and the preparation methods of the bioactive glasspowder used in this example are the same as those in ImplementationExample 2.

The bioactive glass powder (granularity at 150-200 microns) is blendedwith PEG powder (granularity at 200-300 microns) at the mass ratio of40:60. Polyvinyl alcohol solution (concentration at 6%) is added toserve as the adhesive and mixed. This mixture is dry-pressed under apressure of 14 MPa, and pellets of the macroporous materials areobtained by removal from the mold. The pellets are first processed at400° C. to remove organics, and then sintered at 800° C. to obtain thesaid macroporous materials with a compressive strength at approx. 1.5MPa and porosity at about 65%. After being immersed in Simulated BodyFluids for 3 days, the degradation rate of the macroporous glassmaterial is 38% (calculated based on the mass percent of Si releasing).

It is understood and contemplated that equivalents and substitutions forcertain elements and steps set forth above may be obvious to thoseskilled in the art, and therefore the true scope and definition of theinvention is to be as set forth in the following claims.

We claim:
 1. A method of manufacturing a resorbable, macroporousbioactive glass scaffold comprising the steps of: creating a mixtureconsisting essentially of by mass percent about 24-45% CaO, 34-50% SiO₂,0-25% Na₂O, 5-17% P₂O₅, 0-5% MgO and 0-1% CaF₂; melting the mixture at atemperature ranging from about 1380° C. to 1480° C.; cooling, crushingand sieving the mixture to obtain glass powders having granularity ofgreater than 100 microns and up to 300 microns; adding into the glasspowders by mass percent about 20-70% of at least one pore forming agentand about 1-5% of a 5-10% solution of polyvinyl alcohol adhesive to forma composition; pressing the composition of the glass powders, the atleast one pore forming agent and the polyvinyl alcohol adhesive intodyes to produce shaped pellets under a pressure of approximately 2-20MPa; and sintering the pellets under temperatures ranging from about750° C. to about 900° C. for a period of about 1-5 hours, therebymanufacturing a resorbable, macroporous bioactive glass scaffold havinginterconnected pores.
 2. The method of claim 1, wherein the granularityof the pore forming agents is chosen to be between about 50-600 microns.3. The method of claim 1, wherein the at least one pore forming agent ischosen from the group of pore forming agents consisting of polyethyleneglycol, polyvinyl alcohol, paraffin, and polystyrene-divinylbenzene. 4.A method of manufacturing a resorbable, macroporous bioactive glassscaffold comprising the steps of: creating a mixture consistingessentially of, by mass percent about 24-45% CaO, 34-50% SiO₂, 0-25%Na₂O, 5-17% P₂O₅, 0-5% MgO and 0-1% CaF₂; melting the mixture at atemperature ranging from about 1380° C. to about 1480° C.; cooling,crushing and sieving the mixture to obtain glass powders havinggranularity of about 150-300 microns; adding into the 150-300 micronglass powders by mass percent about 20-70% of at least one pore formingagent and about 1-5% of a 5-10% solution of polyvinyl alcohol adhesiveto form a composition; pressing the composition of the 150-300 micronglass powders, the at least one pore forming agent and the polyvinylalcohol adhesive into dyes to produce shaped pellets under a pressure ofabout 2-20 MPa; and sintering the pellets under temperatures rangingfrom about 750 to about 900° C. for a period of about 1-5 hours, therebymanufacturing a resorbable, macroporous bioactive glass scaffold havinginterconnected pores.
 5. The method of claim 4, wherein the granularityof the pore forming agents is chosen to be between about 50-600 microns.6. The method of claim 4, wherein the at least one pore forming agent ischosen from the group of pore forming agents consisting of polyethyleneglycol, polyvinyl alcohol, paraffin, and polystyrene-divinylbenzene. 7.The method of claim 1, wherein the manufactured resorbable, macroporousbioactive glass scaffold has porosity in the range from 40% to 80%. 8.The method of claim 1, wherein the glass powders in the manufacturedresorbable, macroporous bioactive glass scaffold have pore size from 50to 600 microns.
 9. The method of claim 1, wherein the manufacturedresorbable, macroporous bioactive glass scaffold has a compressivestrength at 1-16 MPa.
 10. The method of claim 1, wherein themanufactured resorbable, macroporous bioactive glass scaffold is capableof releasing soluble silicon ions with precipitation of bone-likehydroxyl-apatite crystallites on a surface of the scaffold, followingimmersion into simulated body fluids (SBF).
 11. The method of claim 1,wherein the manufactured resorbable, macroporous bioactive glassscaffold has a degradation rate of approximately 2-30% followingimmersion into simulated body fluids (SBF) for 5 days.
 12. The method ofclaim 4, wherein the manufactured resorbable, macroporous bioactiveglass scaffold has porosity in the range from 40% to 80%.
 13. The methodof claim 4, wherein the glass powders in the manufactured resorbable,macroporous bioactive glass scaffold have pore size from 50 microns to600 microns.
 14. The method of claim 4, wherein the manufacturedresorbable, macroporous bioactive glass scaffold has a compressivestrength at 1-16 MPa.
 15. The method of claim 4, wherein themanufactured resorbable, macroporous bioactive glass scaffold is capableof releasing soluble silicon ions with precipitation of bone-likehydroxyl-apatite crystallites on a surface of the scaffold, followingimmersion into simulated body fluids (SBF).
 16. The method of claim 4,wherein the manufactured resorbable, macroporous bioactive glassscaffold has a degradation rate of approximately 2-30% followingimmersion into simulated body fluids (SBF) for 5 days.